Fluorescent Ca2+ indicators are indispensable tools for studying spatiotemporal fluctuations of intracellular free Ca2+ concentration ([Ca2+]i). Ca2+ is an ubiquitous second messenger involved in numerous intracellular signaling cascades. Biological Ca2+ signals gain their specificity from operating at different temporal, spatial, and concentration scales. Temporally, Ca2+ transients cover the submillisecond to hour scale. Confined transients microdomains coexist with large-scale fluctuations which propagate through multicellular networks that extend over hundreds of micrometers. Cellular transients signals cover concentrations from near ˜100 nM for the basal free [Ca2+]i of most mammalian cells to >100 μM at the peak of Ca2+ microdomains. Thus, depending on the specific Ca2+ signal investigated, Ca2+ indicators with different affinity for Ca2+ binding (KD,Ca) are required as fluorescent reporters.
Calcium indicators are used for imaging in neurosciences, in virology, in cardiology.
The fast on-rate for Ca2+ binding and high selectivity for Ca2+ over Mg2+ has made from BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) the most popular Ca2+ chelator used in the synthesis of chemical Ca2+ indicators (Tsien, R. Y., Biochemistry, 1980, 19, 2396). A broad range of indicators has been synthesized by linking or integrating BAPTA to various fluorophores.
Upon binding of Ca2+ in the chelating moiety BAPTA, the optical properties of the fluorophore are affected in a detectable way and this change may be correlated with the presence of Ca2+ according to a defined standard. This is based on the “PET effect” (Photoinduced Electron Transfert) from the BAPTA ionophoric moiety to the fluorophore moiety, which leads to a decrease in the relative fluorescence intensity and the fluorescence decay time of the fluorophore. By the binding of Ca2+ to BAPTA, the PET effect may be partly or totally inhibited, so that there is an increase in the fluorescence of the fluorophore moiety. Hence the concentration of Ca2+ can be deduced by measuring the change in fluorescence properties, i.e. fluorescence intensity and/or fluorescence decay time.
Most Ca2+ indicators combine BAPTA with fluorescein derivatives and hence emit yellow/green fluorescence (Kao et al., J. Biol. Chem., 1989, 264, 8179; Thomas et al., Cell Calcium, 2000, 28, 213). However, the increasing use of cells transfected with fluorescent proteins (FPs), especially eGFP (enhanced Green Fluorescent Protein), of FP-expressing transgenic mice for targeting identified subpopulations of cells, together with the advent of optical techniques for purposes other than imaging, require the development of new genetically encoded and chemical Ca2+ probes.
Green or yellow FP tags are the most common chromophores used for Ca2+ indicators. GECO-R is the sole red-emitting genetically encoded Ca2±-sensor (Zhao et al, Science's STKE, 2011, 333, 1888) whereas other FP-based Ca2+ indicators remain limited to green. The demand for longer-wavelength and higher signal-to-noise chemical Ca2+ indicators is accentuated by the recent trend toward all-optical manipulation and recording. Photopharmacology, photochemical uncaging, and optogenetics all use near-ultraviolet or short visible wavelengths that further restrain the part of the visible spectrum available for Ca2+ imaging.
Taken together, to be valuable for biological Ca2+ imaging, new Ca2+ probes should be bright, operate in spectral windows outside the yellow/green, and have a tunable KD,Ca.
The main reason for the dominance of fluorescein as fluorophore is its very favorable photo-physical properties (high absorptivity, large quantum yield, and an excitation maximum close to the 488 nm laser line). The X-Rhodamine chromophore is similarly bright as fluorescein, but is red-shifted in both excitation (574 nm) and emission (600 nm) (it is not pH sensitive in the biological pH range and is photostable) and thus presents a suitable alternative fluorophores for indicator design.
Calcium indicators based on rhodamine as fluorophore and BAPTA as Ca2+ chelate are for example described in patent EP0 314 480, enabling to work at long wavelengths. Even if affinity for Ca2+ is interesting, ranging from 370 nM to 2.3 μM, these indicators present the drawback to have a low quantum yield, i.e. a small or negligible shift to absorbance, excitation or emission wavelengths upon Ca2+ binding.
The Applicant described a first family of red emitting calcium indicators based on X-Rhodamine as fluorophore and BAPTA as Ca2+ chelate (Scheme 1): Calcium Rubies (CaRubies) (Gaillard et al., Org. Lett., 2007, 9(14), 2629-2632; Collot, M. et al. JACS, 2012, 134, 14923-14931), which had the additional feature of bearing an azido side arm suitable for functionalization by click chemistry (Kolb, H. C. et al. Angew Chem Int Edit 2001, 40, 2004).

Although CaRubies exhibit good spectral properties, two-photon imaging capabilities and multicolor imaging using optogenetics, their dissociation constants ranged from 3.4 to 21.6 μM which was not ideal for the detection of small [Ca2+] transients in biological tissue.
There is therefore a need for new bright red emitting Ca2+ indicators having a tunable affinity for calcium ranging from the submicromolar range to micromolar range. Especially, there is a need for a series of calcium indicators having increasing affinities, the more sensible calcium indicator of the series having an affinity of less than 300 nM.
The present invention relates to new red emitting Ca2+ indicators comprising a rhodamine moiety and a chelating moiety derived from BAPTA. Especially, the invention relates to a compound of formula I
                wherein Z, R1, R2, R3, R4, m, W, L, Y, R5, R6, R7, R8 and X are as defined below.        
Depending on the substituents of the red-emitting Ca2+ indicators of the invention, affinity for calcium ranging may be modulated. Advantageously, the red-emitting Ca2+ indicators of the invention have a tunable affinity for calcium ranging from the submicromolar range to micromolar range.
It is noteworthy that the fluorophore (rhodamine derivative) is placed in meta position to the nitrogen of the BAPTA, whereas it is in para in every Ca2+ indicators disclosed in the prior art. This shift from para to meta position was surprisingly shown enabling to obtain higher affinities for Ca2+ while keeping optical properties of the fluorophore and without modifying the efficacy of PET quenching.
Using two-photon microscopy and simultaneous patch-clamp recording, it was evidenced that the red emitting Ca2+ indicators of the invention give signals comparable to commonly used green emitting [Ca2+] probes.
Using high-speed random access microscopy, the Applicant further showed that the red emitting Ca2+ indicators of the invention report [Ca2+] transients with kinetics comparable to commonly used indicators.
In vivo patch-clamp recordings demonstrated that the red emitting Ca2+ indicators of the invention are Ca2+ indicators well suited for a wide range of neuroscience experiments, with a signal quality comparable to previously used high-affinity green emitting probes.
Using the strongly overlapping two-photon excitation spectra of eGFP and of the Ca2+ indicator of the invention, a set of experiments was conducted, which was previously not possible. Especially, the potential of two-channel functional imaging was demonstrated with the red emission and high sensitivity of the indicator of the invention being an ideal match for numerous other indicators emitting in the green-yellow spectral band.
It was also evidenced that dual color imaging is also possible and efficient in vivo with the red emitting Ca2+ indicator of the invention in the presence of eYFP.
As a consequence, the red emitting Ca2+ indicators of the invention are ideal indicators for small intracellular [Ca2+] transients. It was shown that the red emitting calcium indicators of the invention are ideal for both in vitro and in vivo imaging experiments requiring high sensitivity to [Ca2+] changes.
Moreover, the Ca2+ indicators of the invention may be combined with activity indicators emitting in the green-yellow spectral band, to allow multiplexed imaging.
Furthermore, the versatility of the indicators of the invention is further increased since they may be functionalized with numerous molecular tools such as for example an antibody, a benzylguanine (SNAP tag) or a peptide to facilitate specific sub-cellular targeting. Especially, the presence of the functionalizable arm enables to introduce moieties suitable to control the localization of the indicator, to make it enter into the cell and to avoid its accumulation in mitochondria once entered in to the cell. Penetrating forms comprising an ester (such as for example an acetoxymethyl—AM) or a dextran are particularly advantageous. With a dextran functionalization, the indicator remains in the cytoplasm.
Indicators of the invention present the advantage that affinity for Ca2+ is not modified upon functionalization.
The red emitting calcium indicators of the invention further present the advantage to be specific to their intended function and not affected by other biologically important metal ions, such as for example Mg2+, Na+ and K+.
Therefore, the red emitting calcium indicators of the invention are powerful and versatile indicators with tunable calcium affinities.